Methods for assessing liver pathologies

ABSTRACT

The present invention provides a new method for detecting or monitoring a liver disease in a subject that has no indication of any liver pathologies, by measuring the amount of concentration of albumin mRNA in an acellular blood sample from the subject, and then comparing the amount or concentration of albumin mRNA with a standard control.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/879,600, now U.S. Pat. No. 9,051,614, which claims priority to U.S.Patent No. 61/241,709, filed Sep. 11, 2009, the contents of each areincorporated by reference in the entirety.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“80015-942245_SEQ” created Jun. 17, 2015 and containing 3,108 bytes. Thematerial contained in this text file is incorporated by reference in itsentirety for all purposes.

BACKGROUND OF THE INVENTION

Many potentially life-threatening liver diseases affect a significantportion of the human population. One such example is hepatitis B, aliver infection caused by the hepatitis B virus (HBV). It is a majorglobal health problem and the most serious type of viral hepatitis, dueto its potential of causing chronic liver diseases, which may ultimatelylead to cirrhosis of the liver and liver cancer. Worldwide, an estimatedtwo billion people have been infected with the HBV, and more than 350million have chronic liver infections though many are asymptomatic.Hepatitis B is endemic in China and other parts of Asia. Most people inthose regions become infected with HBV during childhood, and 8% to 10%of the adult population are chronically infected. Liver cancer caused byHBV is among the first three causes of death from cancer in men, and amajor cause of cancer in women.

A number of liver function tests have been developed and routinely usedin clinics. For example, a patient's blood sample may be tested for thelevel of alanine transaminase (ALT), aspartate transaminase (AST),alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin, orgamma glutamyl transpeptidase (GGT) for the purpose of assessing liverfunction. However, because of the high prevalence of liver diseases andthe vital importance of early detection and treatment, especially inview of the fact that most liver diseases show only mild symptomsinitially, there exists a need for new and more sensitive methods thatwould allow early diagnosis of liver diseases. This invention fulfillsthis and other related needs.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for detecting aliver disease in a subject who has no indication of a liver disease, forexample, the person never had abnormal alanine aminotransferase (ALT)test results. The method includes these steps: (a) determining theamount or concentration of albumin mRNA in an acellular blood sampletaken from the subject; and (b) comparing the amount or concentration ofalbumin mRNA from step (a) with a standard control. An increase in theamount or concentration obtained from step (a) when compared to thestandard control indicates the presence of a liver disease in thesubject. Whereas the amount or concentration of albumin mRNA from step(a) is substantially the same as the standard control, the subject isthen deemed to be free of a liver disease.

In some embodiments, the subject being tested is a person not at risk ofdeveloping a liver disease. Some examples of the liver diseases to betested for include fatty liver disease such as nonalcoholic fatty liverdisease, cirrhosis, liver fibrosis, or hepatitis (e.g., hepatitis A, B,or C). Other examples include Wilson disease, hemochromatosis, alpha1-antitrypsin deficiency, or glycogen storage disease. In someembodiments, hepatocellular carcinoma, especially hepatocellularcarcinoma occurred in a post-liver transplant patient, is excluded fromthe list of liver diseases being tested for using the method of thisinvention.

In some embodiment, the acellular blood sample is plasma. In otherembodiments, the acellular blood sample is serum.

In some embodiments of the claimed method, step (a) comprisesamplification of the albumin mRNA sequence, such as by a polymerasechain reaction (PCR), including reverse transcriptase (RT)-PCR, digitalPCR, or real-time quantitative PCR. In other embodiments, step (a)comprises mass spectrometry or hybridization to a microarray,fluorescence probe, or molecular beacon.

In some cases, the claimed method may further involve repeating step (a)at a later time using the same type of acellular blood sample from thesubject (e.g., when a serum sample was used in step (a), the repeatedstep would use a second serum sample). When the amount or concentrationfrom original step (a) already indicates the presence of liver disease,an increase in the amount or concentration of albumin mRNA at the latertime (i.e., from the repeated step (a)) as compared to the original step(a) indicates a worsening of the liver disease, whereas a decreaseindicates an improvement of the liver disease.

Similarly, the claimed method may further involve repeating step (a) ata later time using the same type of acellular blood sample from thesubject, when the amount or concentration of albumin mRNA from theoriginal step (a) indicates no liver disease. An increase in the amountor concentration of albumin mRNA at the later time (repeated step (a))as compared to the original step (a) indicates the occurrence of a liverdisease, and a substantially lack of change indicates a physiologicalstate of free of the liver disease.

Frequently, an increase or decrease from the standard control in theclaimed method is by at least 1 standard deviation. In other cases, suchincrease or decrease from the standard control may be by at least 2 oreven 3 standard deviations.

In another aspect, the present invention provides a kit for diagnosing aliver disease in a subject who has no indication of a liver disease, ora person who is not at risk of developing a liver disease. The kitincludes these components: (1) a standard control that provides anaverage amount or concentration of albumin mRNA in a blood sample ofhealthy individuals; and (2) two oligonucleotide primers forspecifically amplifying at least a section of albumin mRNA. Typically,the kit further contains an instruction manual to aid user in practicingthe method of this invention. The kit may also include a polynucleotideprobe for specific hybridization with the albumin coding sequence.

In a further aspect, the present invention can also be embodied in adevice or a system comprising one or more such devices, which is capableof carrying out all or some of the method steps described herein. Forinstance, in some cases, the device or system performs the followingsteps upon receiving an acellular blood sample taken from a subjectbeing tested for detecting a possible liver disease or monitored forchanges in liver condition: (a) determining in sample the amount orconcentration of albumin mRNA; (b) comparing the amount or concentrationwith a standard control value; and (c) providing an output indicatingwhether a liver pathology is present in the subject, or whether there isa change, i.e., worsening or improvement, in the subject's livercondition. In other cases, the device or system of the inventionperforms the task of steps (b) and (c), after step (a) has beenperformed and the amount or concentration from (a) has been entered intothe device. Preferably, the device or system is partially or fullyautomated.

In practicing any of the above mentioned aspects of the presentinvention, the person being tested using the method or kit or device orsystem of this invention may be one who has no indication of a liverdisease, or one who has no known risk of developing a liver disease, orone who has received a liver transplant and shown no symptoms of liverabnormality by conventional testing. In some cases, post-livertransplant patients who previously suffered from hepatocellularcarcinoma and the hepatocellular carcinoma recurred subsequent to livertransplantation may be excluded from the practice of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Box plot of plasma ALB mRNA concentrations. The upper and lowerlimits of the boxes and the lines across the boxes indicate the 75^(th)and 25^(th) percentiles and the median, respectively. The whisker capsindicate the 90^(th) and 10^(th) percentiles. Outliers are illustratedas open circles. The dashed line indicates the cutoff for detectingliver pathologies (835 copies/mL) generated by the ROC analysis. HCC,hepatocellular carcinoma; CHB, chronic hepatitis B.

FIG. 2. Receiver-operating characteristic curve for the detection ofliver pathologies by plasma ALB mRNA quantification.

FIG. 3. Correlation between plasma ALB mRNA and alanine transaminaseconcentrations. HCC, hepatocellular carcinoma; CHB, chronic hepatitis B.

FIG. 4. Correlation between plasma ALB mRNA and serum alpha-fetoproteinin hepatocellular carcinoma patients. The inset encloses those patientswith alpha-fetoprotein (AFP)<20 μg/L but plasma ALB mRNAconcentration>835 copies/mL.

FIG. 5. Mass spectra obtained for the genotyping of the ALB SNP,rs962004. The SNP alleles are resolved by the differences in themolecular masses of the extension products. The positions for peaksrepresenting the unextended primer, the extended A and G alleles,respectively, are as marked. The x-axis depicts the mass measured inDaltons, while the y-axis depicts the intensity of ionic currentmeasured in arbitrary units. The hME assay can be applied to thegenotyping of both DNA and RNA products. Samples that were homozygousfor the A-allele, heterozygous and homozygous for the G-allele are shownin the upper, middle and lower panels, respectively.

FIG. 6. Box plot of plasma ALB mRNA concentrations in participantswithout elevated plasma ALT. The upper and lower limits of the boxes andthe lines across the boxes indicate the 75^(th) and 25^(th) percentilesand the median, respectively. The whisker caps indicate the 90^(th) and10^(th) percentiles. Outliers are illustrated as open circles. Thedashed line indicates the cutoff for detecting liver pathologies (835copies/mL) generated by the ROC analysis. HCC, hepatocellular carcinoma;CHB, chronic hepatitis B.

FIG. 7. Receiver-operating characteristic curve for the detection ofliver pathologies by plasma ALB mRNA quantification in subjects withoutelevated plasma ALT.

FIG. 8. Box plot of plasma ALB mRNA concentrations in healthy controlsand post-liver transplant recipients without elevated plasma ALT. Theupper and lower limits of the boxes and the lines across the boxesindicate the 75^(th) and 25^(th) percentiles and the median,respectively. The whisker caps indicate the 90^(th) and 10^(th)percentiles. Outliers are illustrated as open circles. The dashed lineindicates the cutoff for detecting liver pathologies (835 copies/mL) asused in FIGS. 1 and 6.

FIG. 9. Box plot showing the plasma ALB mRNA concentrations of theStable and Unstable groups at the time of enrollment. The upper andlower limits of the boxes and the lines across the boxes indicate the75^(th) and 25^(th) percentiles and the median, respectively. Thewhisker caps indicate the 90^(th) and 10^(th) percentiles. Outliers areillustrated as open circles. The plasma ALB mRNA cutoff level of 835copies/mL is represented by the dotted line.

FIGS. 10A and 10B. Plasma ALB mRNA concentrations for blood samplestaken over the entire duration of the study for recipients in (FIG. 10A)the Stable group and (FIG. 10B) the Unstable groups. Plasma ALB mRNAlevels of stable and unstable recipients are represented by open symbolswith dashed lines and closed symbols with solid lines respectively. Theplasma ALB mRNA cutoff level of 835 copies/mL is represented by thedotted line.

FIGS. 11A to 11F. Serial measurements of plasma ALB mRNA and ALT amongrecipients with acute liver complications. FIGS. 11A to 11F are data ofserial measurements performed for different patients, each representingone patient. Plasma ALB mRNA concentrations are illustrated by filledcircles with solid lines while ALT activity-levels are illustrated byopen circles with dashed lines. The plasma ALB mRNA cutoff level of 835copies/mL is represented by the dotted line.

FIGS. 12A to 12D. Serial measurements of plasma ALB mRNA and ALT amongrecipients with chronic liver complications. FIGS. 12A to 12D are dataof serial measurements performed for different patients, eachrepresenting one patient. Plasma ALB mRNA concentrations are illustratedby filled circles with solid lines while ALT activity-levels areillustrated by open circles with dashed lines. The plasma ALB mRNAcutoff level of 835 copies/mL is represented by the dotted line.

FIG. 13. Plasma ALB mRNA concentrations in cases with fatty liverdisease and healthy controls. The upper and lower limits of the boxesand the lines across the boxes indicate the 75^(th) and 25^(th)percentiles and the median, respectively. The whisker caps indicate the90^(th) and 10^(th) percentiles. Outliers are illustrated as opencircles.

FIG. 14. Plasma ALB mRNA concentrations in cases with fatty liverdisease diagnosed by liver biopsy (Bx), or diagnosed by magneticresonance spectroscopy imaging (MRI) and healthy controls. The upper andlower limits of the boxes and the lines across the boxes indicate the75^(th) and 25^(th) percentiles and the median, respectively. Thewhisker caps indicate the 90^(th) and 10^(th) percentiles. Outliers areillustrated as open circles. NAFLD, nonalcoholic fatty liver disease.

FIG. 15. Plasma ALT activity-concentrations in the subjects shown inFIG. 14. The dashed line indicates the reference cutoff value. Bx,biopsy; MRI, magnetic resonance spectroscopy imaging. NAFLD,nonalcoholic fatty liver disease.

DEFINITIONS

The term “liver disease,” as used in this application, refers to anyevent or condition that alters normal liver functions in a patient,manifesting its symptoms for any length of time during the patient'slife span. Some examples of a liver disease include liver cancer,cirrhosis, liver fibrosis, fatty liver, non-alcoholic steatohepatitis,toxic or mechanical injury to the liver, viral or bacterial infectionsuch as various kinds of hepatitis (e.g., hepatitis A, B, or C), Wilsondisease, hemochromatosis, alpha 1-antitrypsin deficiency, or glycogenstorage disease. In some cases, hepatocellular carcinoma may be excludedfrom the liver diseases to be diagnosed for the purpose of thisapplication. Liver diseases are often subclassified as acute or chronicdepending on the duration of morbidity. In general, conditions thatpersist for more than 3 months are considered chronic liver diseases andthose lasting less than 3 months are considered acute liver diseases.Acute liver diseases, such as acute hepatitis caused by viral orischemic injury to the liver, usually presents abruptly but oftenassociated with full restoration of normal liver function. Chronic liverdiseases, on the other hand, usually presents insidiously with slowprogression but rarely reverts to complete normal liver function.

As liver diseases have varying underlying causes and clinical symptoms,there are numerous different methods for diagnosing these liverdiseases. Conventional methods generally include the analysis of serumbiochemical markers commonly to assess the level of alanine transaminase(ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), totalbilirubin (TBIL), direct bilirubin, or gamma glutamyl transpeptidase(GGT). Alternatively, imaging of the hepatobiliary system by ultrasound,CT scan or MRI, is used to detect structural abnormalities of the liver.A person who is currently not known to have any of those abnormalities,or was never at any time in the past diagnosed with a chronic liverdisease, or was known to have been recovered from a previous acute liverdisease is a “subject who has no indication of a liver disease.” Oneexample of such a person is one who currently is not expected to have anabnormal alanine aminotransferase (ALT) test result because either thisperson never had an abnormal ALT test result in the past or ALT revertedto normal levels after the last acute liver injury. As carriers ofcertain hepatic viruses, such as HBV, can be asymptomatic and withoutovert liver cellular damage to an extent to cause any detectableabnormalities by the conventional liver function tests, a person knownto be a carrier of any one of such viruses but never had an abnormalliver function test result is also considered as a “subject who has noindication of a liver disease.” A person who not only has no indicationof a liver disease but also does not have an immediate family member(parents or siblings) with indication of a liver disease is consideredone who is not at risk of developing a liver disease.

The ALT test is routinely employed by the medical professionals. It isusually requested if one is experiencing symptoms of liver disease,including jaundice (yellowish skin or eyes), dark urine, nausea,vomiting, or abdominal pain. It may also be requested to help diagnoseinfections of the liver such as viral hepatitis or to monitor patientstaking medications that cause liver-related side effects. The normalrange of ALT levels in the blood is between 5 IU/L to 60 IU/L(International Units per Liter). The normal range for AST levels is 5IU/L to 43 IU/L.

The term “blood” as used herein refers to a blood sample or preparationfrom a subject being tested for a possible liver disease or forassessing the physiological state of the subject's liver. An “acellularblood sample” refers to any fraction of blood from which at least 95% ofall cells present in whole blood have been removed, and encompassesfractions such as serum and plasma as conventionally defined. Bloodsamples obtained from different individuals or from the same individualbut at different time points following the same processing steps arereferred to as “the same type of blood samples.”

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, single nucleotide polymorphisms (SNPs), and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) involved in thetranscription/translation of the gene product and the regulation of thetranscription/translation, as well as intervening sequences (introns)between individual coding segments (exons).

In this application, the terms “polypeptide,” “peptide,” and “protein”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

As used in this application, an “increase” or a “decrease” refers to adetectable positive or negative change in quantity from an establishedstandard control. An increase is a positive change preferably at least2-fold, more preferably at least 5-fold, and most preferably at least10-fold of the control value. Similarly, a decrease is a negative changepreferably at least 50%, more preferably at least 80%, and mostpreferably at least 90% of the control. Other terms indicatingquantitative changes or differences from a comparative basis, such as“more,” “less,” “higher,” and “lower,” are used in this application inthe same fashion as described above. In contrast, the term“substantially the same” or “substantially lack of change” indicateslittle to no change in quantity from the standard control value,typically within ±10% of the standard control, or within ±5%, 2%, oreven less variation from the standard control.

A “polynucleotide hybridization method” as used herein refers to amethod for detecting the presence and/or quantity of a polynucleotidebased on its ability to form Watson-Crick base-pairing, underappropriate hybridization conditions, with a polynucleotide probe of aknown sequence. Examples of such hybridization methods include Southernblotting and Northern blotting.

“Primers” as used herein refer to oligonucleotides that can be used inan amplification method, such as a polymerase chain reaction (PCR), toamplify a nucleotide sequence based on the polynucleotide sequencecorresponding to a gene of interest, e.g., the albumin coding sequence.At least one of the PCR primers for amplification of a polynucleotidesequence is sequence-specific for the sequence.

The term “digital polymerase chain reaction” as used herein refers to arefined version of conventional polymerase chain reaction (PCR) methodsthat can be used to directly quantify and clonally amplify nucleic acidsincluding DNA, cDNA or RNA, such that the amount of target nucleic acidcan be directly quantitatively measured. Digital PCR achieves thisdirect quantitative measurement by capturing or isolating eachindividual nucleic acid molecule present in a sample within manyseparate reaction chambers that are able to localize and concentrate theamplification product to detectable levels. After PCR amplification, acount of chambers containing PCR end-product is a direct measure of theabsolute nucleic acids quantity. The capture or isolation of individualnucleic acid molecules, typically by way of dilution, may be effected incapillaries, microemulsions, arrays of miniaturized chambers, or onnucleic acid binding surfaces. The basic methodology of digital PCR isdescribed in, e.g., Sykes et al., Biotechniques 13 (3): 444-449, 1992.

The term “molecular counting” as used herein refers to any method thatallows quantitative measurement of the number of a molecule or molecularcomplex, often the relative number in the context of other co-existingmolecules or complexes of distinct characteristics. Various methods ofmolecular counting are described in, e.g., Leaner et al., AnalyticalChemistry 69:2115-2121, 1997; Hirano and Fukami, Nucleic Acids SymposiumSeries No. 44:157-158, 2000; Chiu et al., Trends in Genetics 25:324-331,2009; and U.S. Pat. No. 7,537,897.

“Standard control” as used herein refers to a predetermined amount of apolynucleotide sequence, e.g., albumin mRNA, that is present in anestablished sample, e.g., an acellular blood sample. The standardcontrol value is suitable for the use of a method of the presentinvention, to serve as a basis for comparing the amount of albumin mRNAthat is present in a test sample. An established sample serving as astandard control provides an average amount of albumin mRNA that istypical for a particular blood sample (particularly an acellular bloodsample, e.g., serum or plasma) of an average, healthy human without anyliver disease as conventionally defined. A standard control value mayvary depending on the nature of the sample as well as other factors suchas the gender, age, ethnicity of the subjects based on whom such acontrol value is established.

The term “average,” as used in the context of describing a human who ishealthy, free of any liver disease as conventionally defined, refers tocertain characteristics, especially the amount of albumin mRNA found inthe person's blood or any acellular fractions of the blood, e.g., serumor plasma, that are representative of a randomly selected group ofhealthy humans who are free of any liver pathologies. This selectedgroup should comprise a sufficient number of humans such that theaverage amount of albumin mRNA in the blood or blood fraction amongthese individuals reflects, with reasonable accuracy, the correspondingalbumin mRNA amount in the general population of healthy humans. Inaddition, the selected group of humans generally have a similar age tothat of a subject whose blood sample is tested for indication of apotential liver disorder. The preferred age for practicing the presentinvention may vary depends on the liver disease/disorder that is beingscreened for. Moreover, other factors such as gender, ethnicity, medicalhistory are also considered and preferably closely matching between theprofiles of the test subject and the selected group of individualsestablishing the “average.”

The term “amount” as used in this application refers to the quantity ofa polynucleotide sequence of interest, e.g., albumin mRNA, present in asample. Such quantity may be expressed in the absolute terms, i.e., thetotal quantity of the polynucleotide sequence in the sample, or in therelative terms, i.e., the concentration of the polynucleotide sequencein the sample.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The analysis of circulating nucleic acids in plasma offers an avenue fornoninvasive monitoring of a variety of physiological and pathologicalconditions (Lo et al., Ann N Y Acad Sci 2004; 1022:135-139; Chan et al.Ann Clin Biochem 2003; 40:122-30). Numerous applications based on thedetection of circulating cell-free nucleic acids in human plasma,ranging from those for the management of malignancies (Anker et al., IntJ Cancer 2003; 103:149-52), pregnancy-associated conditions (Lo et al.,Nat Rev Genet 2007; 8:71-7), organ transplantation (Lo et al., Lancet1998; 351:1329-30) and trauma (Lo et al., Clin Chem 2000; 46:319-23;Chiu et al., Acta Neurochir Suppl 2005; 95:471-4), have been reported.The fundamental principle underlying these applications relate to theplasma detection of extracellular nucleic acid molecules derived fromdiseased organs. Disease-specific genetic signatures that could beexploited from circulating DNA analysis include the detection ofdisease-related pathogens (Chan et al., Clin Chem 2005; 51:2192-5),disease-specific mutations, sex and polymorphism differences between afetus and its mother or a transplant donor and recipient.

In addition to circulating DNA, cell-free plasma RNA analysis offersanother dimension of opportunity for the development ofpathology-related markers (Lo et al., Ann N Y Acad Sci 2004;1022:135-139; Anker et al., Clin Chem 2002; 48:1210-1). Expressionprofiles unique to an organ or disease could be targeted as the specificnucleic acid signature for plasma detection. Tumor- (Chen et al., ClinCancer Res 2000; 6:3823-6) and placenta-derived RNA species (Ng et al.,Proc Natl Acad Sci USA 2003; 100:4748-53) have been successfullydetected from plasma with potential for disease assessment (Ng et al.,Clin Chem 2003; 49:727-31). The present inventors have explored thepossibility of detecting circulating liver-derived mRNA for theassessment of liver pathologies.

There is much evidence to suggest that circulating DNA and RNA arereleased upon cell death (Jahr et al., Cancer Res 2001; 61:1659-65). Asalbumin is the most abundant protein of the body and is synthesized bythe liver, the inventors hypothesized that ALB mRNA may be detectable inhuman plasma and is possibly a sensitive marker of liver pathologies.Indeed, previous studies reported the detection of ALB mRNA inperipheral whole blood and the peripheral mononuclear cell fraction(Hillaire et al., Gastroenterology 1994; 106:239-42; Kar et al.,Hepatology 1995; 21:403-7; Muller et al., Hepatology 1997; 25:896-9;Barbu et al., Hepatology 1997; 26:1171-5; Gion et al., Hepatology 1998;28:1663-8; Peck-Radosavljevic et al., Liver Transplant Oncology Group. JHepatol 1998; 28:497-503; Wong et al., Br J Cancer 1997; 76:628-33; Wonget al., Cancer Lett 2000; 156:141-9; Bastidas-Ramirez et al., HepatolRes 2002; 24:265.11) of human subjects. However, these studies reporteda mixed level of success with detection rates of blood ALB mRNA below100% from patients with hepatocellular carcinoma (HCC), cirrhosis,hepatitis and healthy controls. Yet, Kudo et al., (J Vet Med Sci 2008;70:993-5) recently reported the presence and correlation of plasma ALBmRNA concentration with hepatic injury in rats.

Blood cells are able to “illegitimately” transcribe genes known to bepredominantly expressed by other cell types (Lambrechts et al., AnnOncol 1998; 9:1269-76) and the present inventors have previouslydemonstrated that blood cells are the major contributors of plasmanucleic acids (Lui et al., Clin Chem 2002; 48:421-7). Therefore theinventors first aimed to determine whether plasma or whole blood ALBmRNA were derived from the liver, and second, after confirming the liverorigin of plasma ALB mRNA, it was determined whether quantitativeaberrations could be detected in a variety of liver pathologies. Inorder to achieve the first aim, a previously described RNA-singlenucleotide polymorphism (SNP) strategy (Lo et al., Nat Med 2007;13:218-23; Chan et al., Clin Chem 2007; 53:1874-6) was used to genotypeALB mRNA molecules found in the circulation of recipients of liver orbone marrow transplantations from donors who were genotypicallydifferent for the targeted ALB coding SNP.

II. General Methodology

Practicing this invention utilizes routine techniques in the field ofmolecular biology. Basic texts disclosing the general methods of use inthis invention include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Protein sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage and Caruthers, Tetrahedron Lett.22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purificationof oligonucleotides is performed using any art-recognized strategy,e.g., native acrylamide gel electrophoresis or anion-exchange highperformance liquid chromatography (HPLC) as described in Pearson andReanier, J. Chrom. 255: 137-149 (1983).

The sequence of a genetic marker or genomic sequence used in thisinvention, e.g., the polynucleotide sequence of the albumin gene, andsynthetic oligonucleotides (e.g., primers) can be verified using, e.g.,the chain termination method for sequencing double-stranded templates ofWallace et al., Gene 16: 21-26 (1981).

III. Acquisition of Blood Samples and Extraction of mRNA

The present invention relates to analyzing the amount of albumin mRNAfound in a person's blood, especially an acellular blood sample, as anon-invasive means to detect the presence and/or to monitor the progressof a liver disease or disorder. Thus, the first steps of practicing thisinvention are to obtain a blood sample from a test subject and extractmRNA from the sample.

A. Acquisition of Blood Samples

A blood sample is obtained from a person to be tested or monitored for aliver condition or disorder using a method of the present invention.Collection of blood from an individual is performed in accordance withthe standard protocol hospitals or clinics generally follow. Anappropriate amount of peripheral blood, e.g., typically between 5-50 ml,is collected and maybe stored according to standard procedure prior tofurther preparation.

B. Preparation of Blood Samples

The analysis of albumin mRNA found in a patient's blood sample accordingto the present invention may be performed using, e.g., the whole blood,or more often in an acellular sample such as serum, or plasma. Themethods for preparing serum or plasma from maternal blood are well knownamong those of skill in the art. For example, a subject's blood can beplaced in a tube containing EDTA or a specialized commercial productsuch as Vacutainer SST (Becton Dickinson, Franklin Lakes, N.J.) toprevent blood clotting, and plasma can then be obtained from whole bloodthrough centrifugation. On the other hand, serum may be obtained with orwithout centrifugation-following blood clotting. If centrifugation isused then it is typically, though not exclusively, conducted at anappropriate speed, e.g., 1,500-3,000×g. Plasma or serum may be subjectedto additional centrifugation steps before being transferred to a freshtube for RNA extraction. Any other method of producing an acellularsample from the whole blood is also appropriate for the purpose of thisinvention, so long as the method generates an acellular blood samplethat is substantially cell-free, e.g., having removed at least 90%, 95%,98%, or 99% or more of all cells originally present in the whole bloodsample.

C. Extraction and Quantitation of RNA

There are numerous methods for extracting mRNA from a biological sample.The general methods of mRNA preparation (e.g., described by Sambrook andRussell, Molecular Cloning: A Laboratory Manual 3d ed., 2001) can befollowed; various commercially available reagents or kits, such asTrizol reagent (Invitrogen, Carlsbad, Calif.), Oligotex Direct mRNA Kits(Qiagen, Valencia, Calif.), RNeasy Mini Kits (Qiagen, Hilden, Germany),and PolyATtract® Series 9600™ (Promega, Madison, Wis.), may also be usedto obtain mRNA from a biological sample from a female test subject.Combinations of more than one of these methods may also be used.

It is essential that all contaminating DNA be eliminated from the RNApreparations. Thus, careful handling of the samples, thorough treatmentwith DNase, and proper negative controls in the amplification andquantification steps should be used.

1. PCR-Based Quantitative Determination of mRNA Level

Once mRNA is extracted from a sample, the amount of albumin mRNA may bequantified. The preferred method for determining the mRNA level is anamplification-based method, e.g., by polymerase chain reaction (PCR).

Prior to the amplification step, a DNA copy (cDNA) of the albumin mRNAmust be synthesized. This is achieved by reverse transcription, whichcan be carried out as a separate step, or in a homogeneous reversetranscription-polymerase chain reaction (RT-PCR), a modification of thepolymerase chain reaction for amplifying RNA. Methods suitable for PCRamplification of ribonucleic acids are described by Romero and Rotbartin Diagnostic Molecular Biology: Principles and Applications pp.401-406; Persing et al., eds., Mayo Foundation, Rochester, Minn., 1993;Egger et al., J. Clin. Microbiol. 33:1442-1447, 1995; and U.S. Pat. No.5,075,212.

The general methods of PCR are well known in the art and are thus notdescribed in detail herein. For a review of PCR methods, protocols, andprinciples in designing primers, see, e.g., Innis, et al., PCRProtocols: A Guide to Methods and Applications, Academic Press, Inc.N.Y., 1990. PCR reagents and protocols are also available fromcommercial vendors, such as Roche Molecular Systems.

PCR is most usually carried out as an automated process with athermostable enzyme. In this process, the temperature of the reactionmixture is cycled through a denaturing region, a primer annealingregion, and an extension reaction region automatically. Machinesspecifically adapted for this purpose are commercially available.

Although PCR amplification of the target mRNA is typically used inpracticing the present invention. One of skill in the art willrecognize, however, that amplification of these mRNA species in amaternal blood sample may be accomplished by any known method, such asligase chain reaction (LCR), transcription-mediated amplification, andself-sustained sequence replication or nucleic acid sequence-basedamplification (NASBA), each of which provides sufficient amplification.More recently developed branched-DNA technology may also be used toquantitatively determining the amount of mRNA markers in maternal blood.For a review of branched-DNA signal amplification for directquantitation of nucleic acid sequences in clinical samples, see Nolte,Adv. Clin. Chem. 33:201-235, 1998.

2. Other Quantitative Methods

The albumin mRNA can also be detected using other standard techniques,well known to those of skill in the art. Although the detection step istypically preceded by an amplification step, amplification is notrequired in the methods of the invention. For instance, the mRNA may beidentified by size fractionation (e.g., gel electrophoresis), whether ornot proceeded by an amplification step. After running a sample in anagarose or polyacrylamide gel and labeling with ethidium bromideaccording to well known techniques (see, e.g., Sambrook and Russell,supra), the presence of a band of the same size as the standardcomparison is an indication of the presence of a target mRNA, the amountof which may then be compared to the control based on the intensity ofthe band. Alternatively, oligonucleotide probes specific to albumin mRNAcan be used to detect the presence of such mRNA species and indicate theamount of mRNA in comparison to the standard comparison, based on theintensity of signal imparted by the probe.

Sequence-specific probe hybridization is a well known method ofdetecting a particular nucleic acid comprising other species of nucleicacids. Under sufficiently stringent hybridization conditions, the probeshybridize specifically only to substantially complementary sequences.The stringency of the hybridization conditions can be relaxed totolerate varying amounts of sequence mismatch.

A number of hybridization formats well known in the art, including butnot limited to, solution phase, solid phase, or mixed phasehybridization assays. The following articles provide an overview of thevarious hybridization assay formats: Singer et al., Biotechniques 4:230,1986; Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson,In situ Hybridization, Wilkinson ed., IRL Press, Oxford UniversityPress, Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: APractical Approach, IRL Press, 1987.

The hybridization complexes are detected according to well knowntechniques. Nucleic acid probes capable of specifically hybridizing to atarget nucleic acid, i.e., the mRNA or the amplified DNA, can be labeledby any one of several methods typically used to detect the presence ofhybridized nucleic acids. One common method of detection is the use ofautoradiography using probes labeled with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P, orthe like. The choice of radioactive isotope depends on researchpreferences due to ease of synthesis, stability, and half lives of theselected isotopes. Other labels include compounds (e.g., biotin anddigoxigenin), which bind to antiligands or antibodies labeled withfluorophores, chemiluminescent agents, and enzymes. Alternatively,probes can be conjugated directly with labels such as fluorophores,chemiluminescent agents or enzymes. The choice of label depends onsensitivity required, ease of conjugation with the probe, stabilityrequirements, and available instrumentation.

The probes and primers necessary for practicing the present inventioncan be synthesized and labeled using well known techniques.Oligonucleotides used as probes and primers may be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage and Caruthers, Tetrahedron Letts.,22:1859-1862, 1981, using an automated synthesizer, as described inNeedham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168, 1984.Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson andRegnier, J. Chrom., 255:137-149, 1983.

IV. Establishing a Standard Control

In order to establish a standard control for practicing the method ofthis invention, a group of healthy persons free of any liver disease asconventionally defined is first selected. These individuals are withinthe appropriate parameters, if applicable, for the purpose of screeningfor and/or monitoring liver disorders or diseases such as cirrhosis,liver fibrosis, hepatitis and others using the methods of the presentinvention. Optionally, the individuals are of same gender, similar age,or similar ethnic background.

The healthy status of the selected individuals is confirmed by wellestablished, routinely employed methods including but not limited togeneral physical examination of the individuals and general review oftheir medical history.

Furthermore, the selected group of healthy individuals must be of areasonable size, such that the average amount/concentration of albuminmRNA in the blood obtained from the group can be reasonably regarded asrepresentative of the normal or average level among the generalpopulation of healthy people. Preferably, the selected group comprisesat least 10 human subjects.

Once an average value for the albumin mRNA is established based on theindividual values found in each subject of the selected healthy controlgroup, this average or median or representative value or profile isconsidered a standard control. A standard deviation is also determinedduring the same process. In some cases, separate standard controls maybe established for separately defined groups having distinctcharacteristics such as age, gender, or ethnic background.

V. KITS

The invention provides compositions and kits for practicing the methodsdescribed herein to assess the state of liver physiology or pathology ina subject, which can be used for various purposes such as detecting ordiagnosing the presence of a liver disease, and monitoring theprogression of a liver disease in a patient, especially one who has noindication of any liver injury, disorder, or disease.

Kits for carrying out assays for determining albumin mRNA leveltypically include at least one oligonucleotide useful for specifichybridization with the albumin coding sequence or complementarysequence. Optionally, this oligonucleotide is labeled with a detectablemoiety. In some cases, the kits may include at least two oligonucleotideprimers that can be used in the amplification of albumin mRNA by PCR,particularly by RT-PCR.

Typically, the kits also provide instruction manuals to guide users inanalyzing test samples and assessing the state of liver physiology orpathology in a test subject.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1 I. Materials and Methods Participants

Between June 2006 and April 2008, participants were recruited from thePrince of Wales Hospital, Hong Kong, including (i) patients with a rangeof liver complications who attended the Departments of Medicine andTherapeutics, and Clinical Oncology; (ii) patients who previouslyreceived liver transplantation (LT) at the Department of Surgery and thepaired living donors; (iii) patients who received bone marrowtransplantation (BMT) at the Department of Pediatrics; and (iv) healthyindividuals. Ethical approval was obtained from the institutional reviewboard, and informed consent was obtained from all participants orresponsible guardians.

Ten mL of peripheral blood was collected into EDTA-tubes. Either buccalcells or hair follicle cells were also collected from the BMT patients.For those participants who have received cadaveric LT, archived liverbiopsy tissue specimens of deceased donors were retrieved.

Sample Collection and Processing

Immediately after blood collection, samples were kept at 4° C. andprocessed within 4 h. After gently mixing the blood sample, 0.3 mL ofwhole blood was mixed with 0.9 mL of TRIzol LS reagent (Invitrogen,Carlsbad, Calif.). Plasma was harvested by a double-centrifugationprotocol (Chiu et al., Clin Chem 2001; 47:1607-13). Buffy coat wasisolated after the first centrifugation step and re-centrifuged at 230 gfor 5 min at 4° C. to remove any residual plasma. All samples werestored until nucleic acid extraction as described in this section.

ALB Genotyping

A SNP, rs962004, within the coding region of ALB was targeted. By DNAsequencing of ten unrelated individuals, it was found that the SNP has aminor allele frequency of 0.37. Genotyping was performed by a primerextension reaction using a homogenous MassEXTEND (hME) (Sequenom, SanDiego, Calif.) assay. Extension products for each SNP allele woulddemonstrate distinct masses that could be resolved by matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry analysis(See FIG. 5). Details of the SNP analysis can be found in Table 3.

Genotypes of LT recipients and liver donors were determined using buffycoat DNA. For cadaveric LT cases, the ALB genotypes of the deceaseddonors were determined from the archived liver biopsy tissue DNA. ForBMT recipients, their original ALB genotypes were detected from DNA ofbuccal cells or hair follicles. The ALB genotypes of the bone marrowdonors were assessed using the buffy coat DNA of recipients after BMT.Genotypes of ALB mRNA in plasma or whole blood of the transplantationrecipients were determined using the same genotyping assay on reversetranscribed ALB mRNA.

Quantification of ALB mRNA Transcript in Plasma

One-step reverse transcription—quantitative real-time PCR (RT-qPCR) wasused to measure the plasma ALB mRNA concentrations. The intron-spanningassay for ALB mRNA quantification was designed to amplify a 78-bp ALBamplicon across exon 1 and exon 2 at the 5′ region (GenBank AccessionNo. NM_(—)000477.3) and the sequences are summarized in Table 4.Calibration curves for absolute ALB mRNA quantification were prepared bysubjecting serial dilutions of HPLC-purified single-stranded syntheticDNA oligonucleotides (Sigma-Proligo, Singapore) specifying the targetedALB amplicon (Wong et al., Clin Chem 2005; 51:1786-95), withconcentrations ranging from 3 copies to 3×10⁶ copies per well ofreaction. The amplification of ALB mRNA was monitored by an ABI Prism7900 Sequence Detector (Applied Biosystems, Foster City, Calif.) and theSequence Detection Software version 2.2 (Applied Biosystems). The medianPCR efficiency was 88.3% (SD: 6%, range: 81.1%-98.8%) calculated fromthe calibration curves with a median slope of −3.64 (SD: 0.18, range:3.35-3.88), y-intercept at 40.8 (SD: 1.86, range: 38.1-43.5) andcorrelation coefficient of 0.9958 (SD: 0.0026, range: 0.9905-0.9986).Absolute concentrations of ALB mRNA in plasma were expressed ascopies/mL.

Assessment of Liver Function

Plasma analysis for albumin, total bilirubin, alkaline phosphatase,alanine transaminase (ALT) and alpha-fetoprotein was performed by theChemical Pathology laboratory of the Prince of Wales Hospital, HongKong, using a DPE Modular Analytics system (Roche Diagnostics).Hepatitis B virus (HBV) DNA was quantified in serum of patients withchronic hepatitis B (CHB) infection as previously described (Chan etal., J Med Virol 2002; 68:182-7; Loeb et al., Hepatology 2000;32:626-629). Concentrations>10,000 copies/mL was considered as evidencefor active viral replication (Chan et al., B. J Clin Microbiol 2003;41:4693-5).

Statistical Analysis

Statistical analyses were performed using the SPSS version 15.0 software(SPSS Inc., Chicago, Ill.). Plasma ALB mRNA concentrations were comparedbetween patient and control groups by the Kruskal-Wallis H test andDunn's test as appropriate. Correlations between plasma ALB mRNAconcentrations and other parameters were determined by the Spearman'srank correlation. A p value of less than 0.05 was considered asstatistically significant and all probabilities were two tailed. Anoutlier was identified when the plasma ALB mRNA concentration of thesample was greater than 3 standard deviations from the mean of thecorresponding group. ROC curve was constructed to determine the areaunder the curve (AUC). Sensitivity and specificity were calculated atthe optimal plasma ALB mRNA concentration cutoff point fordistinguishing patients with liver complications from healthyindividuals.

Sample Collection and Preparation

To harvest plasma, each blood sample was first centrifuged at 1 600 gfor 10 min at 4° C. (Centrifuge 5810R, Eppendorf, German). Thesupernatants were carefully transferred into plain polypropylene tubesand re-centrifuged at 16 000 g for 10 min at 4° C. (Centrifuge 5417R,Eppendorf) (Chiu et al., Clin Chem 2001; 47:1607-13). The plasma wasthen transferred into fresh polypropylene tubes without disturbing theunderlying pellet. Every 1.6 mL of plasma was then mixed with 4.8 mL ofTRIzol LS reagent (Invitrogen) and stored at −80° C. until extraction(Heung et al., Prenat Diagn 2009; 29:277-9).

RNA Extraction

Each plasma-TRIzol LS mixture was thawed and mixed with 1.28 mL ofchloroform. The RNA lysate was separated into different phases bycentrifugation at 12 000 g for 15 min at 4° C. The aqueous layer wasthen carefully transferred to fresh polypropylene tubes. For RNAprecipitation, one volume of 700 mL/L ethanol was added to one volume ofthe aqueous layer. The mixture was applied to a spin column of theRNeasy Mini Kit (Qiagen, Valencia, Calif.) and processed according tothe manufacturer's protocols. Total RNA was eluted in 50 μl ofRNase-free water. All RNA samples were pre-treated with AmplificationGrade Deoxyribonuclease I (Invitrogen) according to the manufacturer'sinstructions and then stored at −80° C. until use.

DNA Extraction

DNA extraction of buffy coat, buccal cells, hair follicle cells andparaffinized liver biopsied tissue was performed either with the QIAampBlood Mini Kit (Qiagen) or QIAamp DNA Mini Kit (Qiagen) as appropriateand recommended by the manufacturer. All DNA samples were eluted in 50μL of double distilled water and then be stored at −20° C. until use.

RNA-SNP Analysis by MassARRAY™ Homogenous MassEXTEND™ (hME) AssayFollowed by Mass Spectrometric Detection

Reverse Transcription—

The RNA samples were reverse transcribed by a thermostable avian reversetranscriptase (ThermoScript™ Reverse Transcriptase, Invitrogen) with agene-specific primer (Integrated DNA Technologies; See Table 3) in 0.5μM of final concentration at 55° C. for 1 h.

PCR Amplification—

For the amplification of reverse transcribed cDNA, each reactioncontained 0.6× HotStar Taq PCR buffer with 0.9 mM MgCl₂ (Qiagen), 25 μMeach of dATP, dGTP, and dCTP, 50 μM dUTP (Applied Biosystems). For theamplification of DNA, each reaction contained 1× HotStar Taq PCR bufferwith 1.5 mM MgCl₂ (Qiagen), an additional 1 mM MgCl₂ (Qiagen), 50 μMeach of dATP, dGTP, and dCTP, 100 μM dUTP (Applied Biosystems). For allPCRs, forward and reverse primers (Integrated DNA Technologies,Coralville, Iowa; See Table 4) were at 200 nM and HotStar Taq Polymerase(Qiagen) was at 0.5 U of final concentration. The PCR was initiated at95° C. for 15 min, followed by denaturation at 95° C. for 20 s,annealing at 56° C. for 30 s, extension at 72° C. for 1 min for 45cycles, and, finally, incubation at 72° C. for 3 min.

Base Extension—

PCR products were subjected to shrimp alkaline phosphatase (SAP)treatment with 0.6 U of shrimp alkaline phosphatase (Sequenom), 0.34 μlof MassARRAY™ Homogenous MassEXTEND™ (hME) buffer (Sequenom), and 3.06μl of water. The mixture was incubated at 37° C. for 40 min followed by85° C. for 5 min to remove excess dNTPs. Genotyping was performed withhME assays (Sequenom). For the RNA SNP genotyping, 9 μl of baseextension reaction cocktail containing 1.2 μM extension primer(Integrated DNA Technologies; See Table 3), 1.15 U of Thermosequenase(Sequenom), and 64 μM each of ddATP, ddCTP and dGTP (Sequenom) wereadded to 5 μL of the SAP-treated PCR products. For the DNA SNPgenotyping, 4 μL of base extension reaction cocktail containing 0.771 μMextension primer (Integrated DNA Technologies; see Table I), 1.15 U ofThermosequenase (Sequenom), and 64 μM each of ddATP, ddCTP and dGTP(Sequenom) were added to 10 μL of the SAP-treated PCR products. Thereaction conditions were 94° C. for 2 min, followed by 85 cycles of 94°C. for 5 s, 52° C. for 5 s, and 72° C. for 5 s for RNA-SNP genotyping.For DNA-SNP genotyping 75 cycles of the same thermal profile was usedfor the primer extension reaction.

Liquid Dispensing and MALDI-TOF MS Data Analysis—

The final base extension product was cleaned up by the addition of 12 mgof Clean Resin (Sequenom) and 24 μL of water. The mixtures were mixed ina rotator for 20 min. After centrifugation at 361 g for 5 min, 10 nL ofreaction solution was dispensed onto a SpectroCHIP (Sequenom) by aMassARRAY Nanodispenser S (Sequenom). A MassARRAY Analyzer Compact MassSpectrometer (Bruker, Madison, Wis.) was used for data acquisition fromthe SpectroCHIP. Mass spectrometric data were automatically importedinto a MassARRAY Typer (Sequenom) database for analysis.

Detection of ALB mRNA Transcript in Plasma by Real-Time Quantitative PCR

The assay for ALB mRNA quantification was designed using Primer Expressv2.0 (Applied Biosystems). The forward primer and reverse primer werelocated in exon 1 and exon 2, respectively and were synthesized byIntegrated DNA Technologies. The fluorogenic probe (Applied Biosystems,Foster City, Calif.) spanned the junction between exons 1 and 2 toprevent the amplification of contaminating genomic DNA (See Table 4). Insilico specificity screen and sequence alignment were performed toensure that the amplicon was specific to the targeted location on ALBand had no splice variant.

The reverse transcription—quantitative real-time PCR reactions were setup manually according to the manufacturer's instructions (TaqMan EZRT-PCR Core Reagents; Cat. No. N8080236; Applied Biosystems) in areaction volume of 25 μL. The final concentrations of each component perreaction were as follows: the EZ buffer in 1×, manganese acetatesolution in 3 mM, dNTPs in 300 μM, AmpErase Uracil N-glycosylase in 0.25unit, forward primer in 400 μM, reverse primer in 400 μM, probe in 200μM, rTth DNA polymerase in 2.5 units and no additive was used. Foramplification, 3 μl of extracted plasma RNA was added into each well ofthe 96-well reaction plates (MicroAmp Optical 96-Well Reaction Plate;Cat No. N8010560; Applied Biosystems). The thermal profile used for theALB mRNA analysis was as follows: the reaction was initiated at 50° C.for 2 min for the included uracil N-glycosylase to act, followed byreverse transcription at 60° C. for 30 min. After a 5-min denaturationat 95° C., 45 cycles of PCR were carried out with denaturation at 94° C.for 20 s and 1 min of annealing/extension at 58° C. Each sample wasanalyzed in duplicate, and the corresponding calibration curve was runin parallel with each analysis.

Multiple water blanks and liver tissue RNA were included in everyanalysis as negative and positive controls, respectively. Samples werealso tested to ensure that they were negative for DNA by substitutingthe rTth polymerase with the AmpliTaq Gold enzyme (Applied Biosystems).No amplification was observed in all negative water blanks and “No-RT”control analysis, indicating the specificity of the assay for mRNA.Specificity of the PCR product was validated by gel electrophoresisanalysis. The limit of detection for ALB analysis was found to be 3copies per reaction well because 98% of 40 duplicate wells [=80 wells intotal], containing 3 copies per reaction well, were successfullydetected with a median (range) threshold cycle of 38.6 (37.8-44.3).Intra-assay CV of the ALB mRNA assay was evaluated by measuring a samplewith ALB mRNA concentration adjusted to 835 copies/mL, which was definedas the cutoff level of plasma ALB mRNA concentration for identifyingpatients with liver disease from healthy controls by the ROC analysis,in 20 duplicate wells on the same reaction plate. The mean ALB mRNAconcentration detected from the replicates was 855 copies/mL with SD of85 copies/mL and CV of 9.97%.

II. Results

Origin of ALB mRNA in Circulation

To determine if ALB mRNA in plasma and whole blood is liver-derived, aRNA-SNP assay was developed to genotype the ALB mRNA molecules found inthe circulation of LT and BMT recipients. The analysis was focused oninformative donor-recipient pairs who were defined as donors who bore adifferent genotype for the interrogated ALB SNP than their correspondingrecipients. After liver transplantation, the genotype corresponding tothat of the donor should be observed for the ALB mRNA molecules found inthe circulation of the recipient if the ALB mRNA were genuinelyliver-derived. Alternatively, if other tissue sources contributed to thepool of circulating ALB mRNA, the ALB genotype of the recipient shouldbe detectable. To demonstrate that hematopoietic cells could be acontaminating source of circulating ALB mRNA, a similar RNA-SNP analysiswas performed for recipients of BMT. Similarly, the circulating ALB mRNAmolecules would exhibit the genotype of the bone marrow donor ifhematopoietic cells contributed ALB mRNA to the circulation.

Twenty-nine (29) LT cases were studied where nine (9) recipientsobtained livers from their living relatives and the remaining fromcadavers. Fifteen (15) of these donor-recipient pairs were deemedinformative by showing distinct ALB genotypes between the donor andrecipient. Table 1 summarizes the genotyping data of the informativedonors and recipients. Among the informative cases, the ALB mRNAgenotypes detected in the plasma of the recipients after transplantationwere different from their original genotypes and corresponded to that ofthe liver donors.

Five of the 20 BMT cases recruited were informative and the genotypingdata are summarized in Table 1. There was no change in the ALB mRNAgenotypes in plasma of the recipients before and after BMT. Thus, thedonor's bone marrow was not a significant contributor of ALB mRNA in therecipient's plasma.

Besides plasma, RNA-SNP analysis was also performed on ALB mRNA in wholeblood collected from the informative LT and BMT recipients aftertransplantation. Twelve (12) informative LT cases and four (4)informative BMT cases consented to this analysis and the genotyping dataare shown in Table 1. Unlike the plasma data, contributions from thebone marrow donors (cases B8 and B10) and the LT recipients (cases L8,L18, L23, L24 and L25) were observed in the whole blood ALB mRNA. Insummary, plasma ALB mRNA was liver-derived while ALB mRNA in whole bloodwas not liver-specific.

Quantitative Analysis of Plasma ALB mRNA

After confirming that plasma ALB mRNA was liver-specific, the inventorsassessed its concentration in plasma obtained from 107 patients with aspectrum of liver complications and 207 healthy individuals. Among thepatients, 35 were confirmed to have HCC, 25 had biopsy-proven livercirrhosis, 24 had CHB with serum HBV DNA concentrations>10,000copies/mL, i.e., active viral replication, and 23 had CHB serum HBV DNAconcentrations<10,000 copies/mL, i.e., inactive viral replication. Allhealthy individuals were tested to be HBsAg negative and had liverfunction test parameters within reference intervals according tostandard plasma biochemical tests.

Information about the demographics, biochemical testing, virologicalinvestigations and the median plasma ALB mRNA concentrations of theparticipants are summarized in Table 2. Plasma ALB mRNA was detected in308 of the 314 participants (98.1%) with negative readings for onepatient and five controls. Concentrations of plasma ALB mRNA of all thegroups are shown in FIG. 1. Plasma ALB mRNA concentrations werestatistically significantly different among the participant groups(P<0.0001, Kruskal-Wallis test). Subgroup analysis showed that patientswith HCC (P<0.0001), liver cirrhosis (P=0.0068) and active CHB(P<0.0001) had statistically significant elevation in plasma ALB mRNAconcentrations when compared to controls. There was no statisticallysignificant difference in plasma mRNA concentrations between controlsand patients with inactive CHB (P=0.4239, Dunn's test). These datafurther support that persons with inactive CHB generally have liverstatus similar to that of healthy controls.

ROC curve analysis was performed to assess if plasma ALB mRNAconcentration would be an effective indicator of liver pathologies (FIG.2). The AUC suggested that the diagnostic efficacy of using plasma ALBmRNA as an indicator of liver pathologies was 92.9%. By using 835copies/mL of plasma ALB mRNA as a cutoff, the sensitivity andspecificity for detecting any one of the assessed liver pathologies were85.5% and 92.8% respectively.

Relationship Between Plasma ALB mRNA Concentrations and ConventionalLiver Function Test Parameters

Considering data from all the study participants (patients andcontrols), plasma ALB mRNA concentration weakly correlated with plasmatotal bilirubin (r=0.133, P=0.018, Spearman's correlation), alkalinephosphatase (r=0.126, P=0.0255) and ALT (r=0.207, P=0.0002; FIG. 3).

Of 107 patients, only 23 (21.5%) patients had elevated ALT levels (>58IU/L), whilst 62 (73.8%) patients had plasma ALB mRNA concentrations>835copies/mL. For the 35 patients with HCC confirmed by liver biopsy, only17 (49%) patients had elevated alpha-fetoprotein levels (>20 μg/L)(Sherman et al., Hepatology 1995; 22:432-8). However, 32 (91.4%) ofthese patients had elevated plasma ALB mRNA concentrations as shown inFIG. 4.

Plasma ALB mRNA Analysis in Subjects with Normal Liver Function

It was remarkable to note that plasma ALB mRNA was elevated in patientswith liver disease but normal plasma ALT or AFP levels. ALT is acustomarily used biochemical marker to indicate hepatocellular damage.These data indicate that ALB mRNA is more sensitive than plasma ALT fordetection of liver diseases. In view of these findings, the data werereanalyzed to only include subjects with normal plasma ALT. Thereference range for normal plasma ALT provided by the testing laboratoryinvolved in this study was <58 U/L.

A total of 291 study participants had normal plasma ALT. Thisrepresented 93% of all participants. Information about the demographics,biochemical testing, virological investigations and the median plasmaALB mRNA concentrations of this subgroup of participants are summarizedin Table 5. Concentrations of plasma ALB mRNA of all the groups withnormal plasma ALT are shown in FIG. 6. Plasma ALB mRNA concentrationswere statistically significantly different among the subgroups(P<0.0001, Kruskal-Wallis test). Further analysis showed that patientswith HCC (P<0.0001), liver cirrhosis (P=0.0094) and active CHB(P<0.0001) had statistically significant elevation in plasma ALB mRNAconcentrations when compared to controls. There was no statisticallysignificant difference in plasma mRNA concentrations between controlsand patients with inactive CHB (P=0.2723, Dunn's test).

ROC curve analysis was performed for this subgroup (FIG. 7). The AUCsuggested that the diagnostic efficacy of using plasma ALB mRNA as anindicator of liver pathologies was 87%. By using 835 copies/mL of plasmaALB mRNA as a cutoff, the sensitivity and specificity for detecting anyone of the assessed liver pathologies were 74% and 93% respectively.

This subgroup analysis further affirms that plasma ALB mRNA analysis isa sensitive marker for the detection of liver pathologies even amongindividuals with normal liver function test, such as normal plasma ALTlevel.

Elevated Plasma ALB mRNA as a Predictor of Future Adverse Outcome

The 29 LT recipients who participated in the genotyping study describedabove were further investigated. Besides genotyping, plasma ALB mRNAconcentration was measured by real-time quantitative PCR as describedabove. Plasma ALT level was also assessed on the same occasion. All ofthe patients was not known to have developed post-LT HCC at the time ofstudy recruitment, unlike the subjects described in Cheung et al.,Transplantation 2008; 85:81-7. The participants were monitored for anyadverse outcomes that required medical attention or hospital admissionfor up to 125 weeks. Five participants did not return for follow-up atthe Prince of Wales Hospital, Hong Kong, and were excluded from thispart of the study. Of the remaining 24 participants, nine developed anadverse outcome other than HCC on a subsequent date as shown in Table 6.Seven of these nine individuals showed elevated plasma ALB mRNA (>835copies/mL) concentration that predated the development of medicalcomplication. Only three of these subjects had an elevated plasma ALTlevel at the time of blood taking. These data indicate that elevatedplasma ALB mRNA concentration is an early predictor of liver-relatedcomplications in post-LT recipients with better performance than plasmaALT. These data indicate that liver-related complications other than HCCcan also be predicted by an elevated ALB mRNA level. Among the 15participants who had not developed liver-related complications, only oneperson (case 14 in Table 6) had a plasma ALB mRNA level>835 copies/mL.The proportion of subjects with elevated plasma ALB mRNA among thosewith and without the development of liver-related complications wasstatistically significantly different (P<0.001, Fisher Exact test).These data indicate that elevated ALB mRNA level is a relativelyspecific early sign and predictor of liver diseases. The extent ofplasma ALB mRNA can be indicative of the severity of the livercomplication, i.e., with prognostic value.

Plasma ALB mRNA Concentration in Individuals Who have Fully Recoveredfrom Prior Liver Diseases

Resumption of ALB mRNA to normal levels after a liver complicationindicates restoration of normal liver function. All of the LT recipientsrequired LT due to conditions that led to severe compromise of theirliver function. Thus, they were treated by LT where the originaldiseased liver was replaced by a healthy liver. For those who did notsubsequently develop any liver-related complications in the post-LTperiod (Table 6), it was interesting to note that the plasma ALB mRNAconcentrations were comparable to the healthy controls shown in FIGS. 1and 6 with no statistically significant difference (P=0.686,Mann-Whitney test) (FIG. 8). These data indicate that despite previousliver insults, after receiving a liver with normal liver function asindicated by the normal plasma ALT and lack of related signs andsymptoms, the plasma ALB mRNA concentration reverts to that of normallevels. Similarly, it is expected that for individuals who recoveredfrom liver injuries as indicated by a normal liver function test andlack of related signs and symptoms but did not receive LT as thetreatment, the plasma ALB mRNA concentration should normalize. Thus, ifplasma ALB mRNA concentration increases at a later stage after recovery,it would be indicative of the development of recurrence or another liverdisease.

III. Discussion

There is much excitement over the possibility of developing blood-basedtools for disease diagnosis and management through the analysis ofcirculating nucleic acids (Chan et al. Ann Clin Biochem 2003; 40:122-30;Anker et al., Int J Cancer 2003; 103:149-52; Lo et al., Nat Rev Genet2007; 8:71-7; Lo et al., Lancet 1998; 351:1329-30; Lo et al., Clin Chem2000; 46:319-23). The detection of circulating RNA offers certainadvantages over that for DNA (Lambrechts et al., Ann Oncol 1998;9:1269-76). As the expression profile between cell types and diseasesare different, tissue- or disease-specific transcripts could beexploited as markers for disease assessment. If a RNA transcript that isunique to a particular organ was selected, the RNA approach may be moregenerally applicable to diseases of that organ and not limited to thefraction of cases harboring specific DNA signatures. Furthermore, ifboth plasma RNA and DNA were derived from the same cell population, thereleased RNA would likely be quantitatively more abundant than that forDNA. This is because multiple copies of a RNA transcript could bepresent in each cell depending on its expression level, while each cellonly contains one diploid genome-equivalent of DNA. Indeed, some cancerresearchers reported that a greater proportion of cancer cases werepositive for the investigated plasma RNA than DNA markers (Anker et al.,Int J Cancer 2003; 103:149-52).

An increasing amount of evidence suggests that liberation of cell-freenucleic acids into plasma from organs or compartments is likely to bedue to cell death (Jahr et al., Cancer Res 2001; 61:1659-65; Fournie etal., Cancer Lett 1995; 91:221-7; Fournie et al., Gerontology 1993;39:215-21). The liver being one of the largest organs of the body, wesuspect that liver expressed RNA, such as ALB, should be detectable inthe peripheral circulation as a result of cell death associated withnormal cell turnover and/or pathological damage. Indeed, studies havereported the presence of circulating ALB mRNA but with varying degreesof success (Cheung et al., Transplantation 2008; 85:81-7). Someresearchers suggested that the blood ALB mRNA originated from malignantor non-malignant hepatocytes that have entered into the peripheralcirculation (Hillaire et al., Gastroenterology 1994; 106:239-42; Kar etal., Hepatology 1995; 21:403-7). However, Muller et al. (Hepatology1997; 25:896-9) reported that peripheral mononuclear cells can beinduced to express ALB mRNA. Indeed, previous reports indicated thatcertain supposedly organ-specific transcripts detectable in thecirculation may in fact be derived from other cell populations, such ashematopoietic cells (Chan et al., Clin Chem 2007; 53:1874-6; Heung etal., PLoS One 2009; 4:e5858) due to illegitimate gene transcription(Lambrechts et al., Ann Oncol 1998; 9:1269-76; Chelly et al., Proc NatlAcad Sci USA 1989; 86:2617-21).

Thus, in this study, the inventors first aimed to confirm if ALB mRNAdetectable in human plasma and whole blood were derived from andspecific to the liver. Donor and recipient pairs of LT or BMT werestudied who were genotypically different for an ALB coding SNP anddetermined the RNA-SNP genotypes in plasma and whole blood. The datademonstrated that the ALB mRNA detected in plasma but not whole bloodwas liver-specific. The data also indicated that ALB mRNA expressed byhematopoietic cells could contribute to the pool of ALB mRNA detected inwhole blood. These findings call for caution in the interpretation ofthe previously reported data on ALB mRNA detection in whole blood.Plasma is preferred over whole blood for future studies on ALB mRNA as abiomarker for liver diseases. To minimize the chance of residual bloodcells contaminating the ALB mRNA molecules in plasma, plasma should beprepared by two centrifugation steps as previously reported.

The inventors then developed a one-step RT-qPCR assay for plasma ALBmRNA quantification. The detection rate for plasma ALB mRNA in our studywas 98.1%. Using two-step RT-qPCR, a recent study investigated the roleof plasma ALB mRNA detection for prediction of HCC recurrence in livertransplant recipients reported a detection rate of 82% (Cheung et al.,Transplantation 2008; 85:81-7). In the discussion section of theirreport, Cheung et al. stated that they believed the positive detectionof plasma ALB mRNA indicated the presence of circulating HCC cells whileLT recipients negative for plasma ALB mRNA suggested the absence of HCCcells in the circulation. Data from the present study, however, showedthat plasma ALB mRNA is detectable in plasma of almost all subjects,including healthy controls. These data indicate that circulating HCCcells are not the sole source of plasma ALB mRNA if at all. Thus, plasmaALB mRNA is more likely to be released into plasma due to any liver celldeath and is useful for the detection of a range of liver diseases notlimited to post-LT HCC. It is believed that the improvement by thepresent inventors in the detection rate for plasma ALB mRNA may berelated to the analytical sensitivity of the one-step RT-qPCR protocoland their targeting of the more 5′ end of the ALB gene. The inventorshave previously reported that circulating mRNA in plasma may not beintact full-length transcripts and showed a 5′ predominance (Wong etal., Clin Chem 2005; 51:1786-95).

It was found that plasma ALB mRNA concentrations were significantlyhigher than controls in patients with HCC, cirrhosis, and active CHB butnot inactive CHB. These data indicate that ALB mRNA is released intoplasma as a result of cell death and may correlate with the degree ofcell death.

By ROC curve analysis, it was found that plasma ALB mRNA measurement wasan attractive means (92.9%) for detecting the presence of liverpathologies. In particular, for the HCC group, alpha-fetoprotein wasonly elevated in 48.6% of the cases while the majority (91.4%) showedelevated ALB mRNA concentrations.

Albeit its diagnostic sensitivity, plasma ALB mRNA is not specifictowards liver pathologies of any particular etiology. However, plasmaALB mRNA concentration bore some correlation with serum ALTactivity-concentration. Plasma ALB mRNA was also found to be elevated inpatients with liver disease but normal ALT levels (FIG. 3). Theseobservations indicate that plasma ALB mRNA is a sensitive marker for thedetection of early stage chronic liver disease where ALTactivity-concentration is often within reference limits.

In summary, experimental data by the present inventors revealed thatplasma but not whole blood ALB mRNA was derived from and specific to theliver. Elevation of plasma ALB mRNA concentrations was observed for arange of liver pathologies and further studies are required toinvestigate its clinical utility in the assessment or management of suchdiseases.

Example 2 Sensitive Detection of Post-Liver TransplantationComplications by Plasma Albumin mRNA Monitoring

In this study, the present inventors showed that significantly elevatedconcentrations of plasma ALB mRNA were associated with chronichepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). Theirdata revealed that plasma ALB mRNA was more sensitive than plasmaalanine transaminase (ALT) activity for the detection of liverpathologies. Long term monitoring of liver function and the earlydetection of hepatic insults are important aspects of the continual careof patients with various liver pathologies, including the post-livertransplantation recipients. Hence, this study was aimed to investigatewhether plasma ALB mRNA measurements offered any value in the care ofpatients known previously to have suffered from liver pathologies, usingrecipients of liver transplantation as an example.

I. Materials and Methods Participants

From June 2006 to July 2009, 24 patients were recruited who previouslyunderwent liver transplantation at the Department of Surgery, the Princeof Wales Hospital, Hong Kong. The post-liver transplantation recipientswere typically seen at the liver transplantation clinic every three tosix months, or more often if clinically indicated. During each visit,physical, biochemical and serological examinations were performed. Theserial analysis of plasma ALB mRNA in this study involved a maximum of 7collections of 6 mL of peripheral blood from a forearm vein within the3-year time period. Ethical approval was obtained from the institutionalreview board and informed consent was obtained from each recipient.

Sample Collection and Processing

The methods of sample collection and processing were as has beendescribed (Chan et al., Clin. Chem. 2010; 56:82-9). In brief, the bloodspecimens collected into EDTA-containing tubes were immediately kept at4° C. and processed within 4 h. Plasma was harvested by adouble-centrifugation protocol (Chiu et al., Clin. Chem. 2001;47:1607-13)). Plasma RNA was extracted by a protocol involving the useof RNeasy Mini Kit (Qiagen, Valencia, Calif.) with TRIzol LS reagent(Invitrogen, Carlsbad, Calif.) (Heung et al., Prenat. Diagn. 2009;29:277-9). Total RNA was eluted in 50 μL of RNase-free water and treatedwith Amplification Grade Deoxyribonuclease I (Invitrogen) according tothe manufacturer's instructions and then stored at −80° C. until use.

Quantification of ALB mRNA Transcript in Plasma

One-step reverse-transcription quantitative real-time polymerase chainreaction (RT-qPCR) was used to measure the plasma ALB mRNAconcentrations. Briefly, the intron-spanning assay for ALB mRNAquantification was designed to amplify a 78-bp ALB amplicon across exon1 and exon 2 at the 5′ region. The reaction was set up using the EZ rTthRNA PCR reagent set (Applied Biosystems, Foster City, Calif.) in areaction volume of 25 μL. Calibration curves for absolute ALB mRNAquantification were prepared by subjecting serial dilutions of HighPerformance Liquid Chromatography-purified single-stranded synthetic DNAoligonucleotides (Sigma-Proligo, Singapore) specifying the targeted ALBamplicon. The lower limit of detection of this assay was 1 copy perreaction with a range of linearity up to 10⁶ copies per reaction. Eachsample was analyzed in duplicate, and the corresponding calibrationcurve was run in parallel with each analysis. The amplification of ALBmRNA was monitored by an ABI Prism 7900 Sequence Detector (AppliedBiosystems) and the Sequence Detection Software version 2.2 (AppliedBiosystems). Absolute concentrations of ALB mRNA in plasma wereexpressed as copies/mL.

Assessment of Liver Function

Plasma analysis for albumin, total bilirubin, alkaline phosphatase(ALP), alanine transaminase (ALT) and alpha-fetoprotein was performed bythe Chemical Pathology laboratory of the pH, using the DPE ModularAnalytics system (Roche Diagnostics, Basel, Switzerland).

Statistical Analysis

Statistical analyses were performed using the SigmaStat for WindowsVersion 3.5 software (Systat Software Inc., Chicago, Ill.). Continuousvariables were expressed as median and range. Plasma ALB mRNAconcentrations and other parameters were compared between groups by theKruskal-Wallis H test and Mann-Whitney U test as appropriate.Correlations between plasma ALB mRNA concentrations and other parameterswere determined by the Spearman's rank correlation. A p-value of lessthan 0.05 was considered as statistically significant and allprobabilities were two tailed.

II. Results

A total of 24 liver recipients, 20 males and 4 females, who had at least3 blood collections for plasma ALB mRNA measurement during the studyperiod, were studied. The liver transplantations were performed at thePWH between 1991 and 2004. Nine were living donor liver transplantationswhile 15 were deceased donor liver transplantations. The indications forliver transplantations included HCC (12 cases), cirrhosis (7 cases),hepatic failure (3 cases), fulminant hepatitis B (1 case) and alcoholiccirrhosis (1 case).

In this study, the 24 recipients were divided into two groups accordingto whether they developed liver-associated clinical sequelae during thestudy period. Fourteen recipients (12 males and 2 females) hadunremarkable course as reflected by their stable clinical condition andbiochemical liver function test profile were classified as the ‘Stable’group. On the other hand, 10 recipients (8 males and 2 females) who hadevidence of single or multiple episodes of liver-associatedcomplications requiring hospitalization or surgical management duringthe study period were classified as the ‘Unstable’ group. None of the 24recipients had HCC recurrence during the study period.

Initial Analyses at the Time of Study Enrollment

At enrollment, the median age of the 24 recipients was 56.2 years old(range, 17-69) and there was no statistically significant difference inthe median ages between the Stable and Unstable groups (Mann-Whitney Utest, P=0.254).

All 14 recipients in the Stable group were deemed to be clinicallystable at the time of enrollment based on an assessment by ahepatologist and the satisfactory biochemical liver function testprofile. For the stable group, the median plasma values of albumin,bilirubin, ALP and ALT levels were 44 g/L (40-49), 15 μmol/L (7-38), 82U/L (59-290) and 32 U/L (10-69), respectively.

Nine of the ten recipients in the Unstable group were clinically stableat the time of enrollment into the study. The one exception, recipientNo. U07, was diagnosed with recurrence of alcoholic cirrhosis and mildchronic rejection at the time of enrollment. The median plasma albumin,bilirubin, ALP and ALT concentrations of the Unstable group were 44 g/L(38-45), 19 μmol/L (10-31), 169 U/L (73-379) and 53 U/L (24-155),respectively. There was no statistically significant difference in theplasma concentrations of albumin (P=0.341) and bilirubin (P=0.538)between the two groups. However, the plasma ALP (P=0.015) and ALT(P=0.035) concentrations of the Unstable group were statisticallysignificantly higher than the Stable group.

The plasma ALB mRNA concentrations of the two groups at the time ofenrollment were shown in FIG. 9. The median plasma ALB mRNAconcentration of the Stable group was 533 copies/mL (69-2,399) whilethat of the Unstable group was 1,540 copies/mL (203-37,524) (P=0.021,Mann-Whitney U test). In their previous study (Chan et al., Clin. Chem.2010; 56:82-9), using receiver-operating characteristic curve analysis,the inventors identified 835 copies/mL as a sensitive and specificcutoff for distinguishing healthy controls from patients with liverpathologies. Adopting the same cutoff for this study, the inventorsfound that 21% ( 3/14) and 70% ( 7/10) of those in the Stable andUnstable groups, respectively, had elevated plasma ALB mRNAconcentrations. The proportion of cases with elevated plasma ALB mRNAconcentrations among the two groups was statistically significantlydifferent (Chi-square P<0.0001). The inventors further studied therelationship between plasma ALB mRNA concentration and the biochemicalliver function test parameters of the 24 recipients. Plasma ALB mRNAconcentration was significantly correlated with the plasmaconcentrations of ALP (Spearman correlation, r²=0.71, P<0.0351) and ALT(r²=0.45, P=0.03), but not with albumin (r²=−0.16, P=0.46) or bilirubin(r²=0.15, P=0.5).

Serial Monitoring of Plasma ALB mRNA

The clinical course of the 24 recipients were followed for a medianduration of 110 weeks. The overall study period of the Stable group, 112weeks (87-160), was comparable with that of the Unstable group, 107weeks (69-159), without any statistically significant difference(P=0.412). During the study period, 105 blood specimens (56 from theStable group and 49 from the Unstable group) were collected for plasmaALB mRNA analysis and all had detectable plasma ALB mRNA

Post-Liver Transplantation Recipients with Stable Clinical Course

Twelve of the 14 recipients (86%) in the Stable group had unremarkableclinical course throughout the study period. The median values of thealbumin, bilirubin, ALP and ALT plasma concentrations of the Stablegroup for all measurements taken during the study were 44 g/L (35-49),15 μmol/L (5-38), 85 U/L (59-290) and 28 U/L (10-158), respectively.Consistent with the first blood sample taken at enrollment, the medianconcentration of plasma ALB mRNA of the Stable group was 418 copies/mL(52-15,320). FIG. 10A is a plot of all plasma ALB mRNA measurementstaken from the Stable group. Only 25% (14/56) of the plasma ALB mRNAmeasurements were above 835 copies/mL. When all plasma ALB mRNAmeasurements from the Stable group were grouped, the 5^(th) and 95^(th)percentile values were 90 copies/mL and 2,400 copies/mL, respectively.The biochemical liver function test profile remained unremarkable on alloccasions for these 12 recipients.

Of the 14 stable recipients, 2 (14%) had an occasional rise in thebiochemical liver function test parameters which returned promptly towithin the normal range without intervention. The plasma ALB mRNAconcentrations from the two recipients measured during those occasionswere 15,320 and 2,399 copies/mL. Yet, the levels returned to 497 and1,044 copies/mL, respectively, upon the subsequent visit. Thecorrelation analysis demonstrated that the plasma ALB mRNAconcentrations measured from the 14 stable recipients during the studyperiod were not significantly correlated to the plasma concentrations ofALP (r²=0.41, P=0.002), but not for albumin (r²=−0.06, P=0.68),bilirubin (r²=−0.19, P=0.16), ALT (r²=0.19, P=0.16).

Post-Liver Transplantation Recipients with Unstable Clinical Course

The 10 recipients in the Unstable group had at least one episode ofliver-associated complication during the study period. Table 7 lists theliver-associated complications developed among the recipients of theUnstable group during the study period. Grouping all the measurementstaken throughout the study period, the median plasma albumin, bilirubin,ALP and ALT concentrations among recipients of the Unstable group were40 g/L (16-46), 23 μmol/L (5-250), 150 U/L (46-601) and 50 U/L (20-413),respectively. The median plasma ALB mRNA level of the Unstable group was2,721 copies/mL (203-61,694), which was 6.5-fold higher than that of theStable group (418 copies/mL). In contrast to the persistently lowconcentrations of plasma ALB mRNA observed among the stable recipients,highly fluctuant or even persistently elevated concentrations of plasmaALB mRNA could be observed in the unstable recipients, as illustrated inFIG. 10B. 78% ( 38/49) of the plasma ALB mRNA measurements from theUnstable group were above 835 copies/mL. The proportion of measurementswith elevated plasma ALB mRNA concentrations in the Stable and Unstablegroups was statistically significantly different (Chi-square P<0.0001).

For the 10 recipients in the Unstable group, six developed acute livercomplications (Cases U01 to U06) and four had chronic livercomplications (Cases U07 to U10). The serial measurements of the plasmaALB mRNA concentrations and the ALT activity-concentrations for theacute and chronic groups were plotted in FIGS. 11A-11F and 12A-12D,respectively.

Unstable Recipients with Acute Liver-Associated Complications

The time of diagnosis of each episode of liver-associated complicationsis marked in the plots within FIGS. 11A-11F and 12A-12D. Among the sixrecipients with documented acute liver-associated complications,elevation in plasma ALB mRNA concentrations (beyond the cutoff value of835 copies/mL) predated the time the diagnosis of the complication wasmade in four cases (FIGS. 11A-11F). In total, nine episodes ofcomplications were documented for cases U01 to U06. The plasma ALB mRNAconcentrations were elevated in all episodes of complications at thetime when the diagnoses were made. In contrast, the plasma ALT activitywas only elevated (>58 U/L) in four of the nine episodes.

Unstable Recipients with Chronic Liver-Associated Complications

Four recipients in the Unstable group had a total of five episodes ofliver-associated complications that were chronic in nature. The plasmaALB mRNA concentrations were elevated at the time of diagnosis exceptwhen case U10 was found to have a liver abscess (FIGS. 12A-12D). PlasmaALT activity was elevated at the time of diagnosis of three of theepisodes.

III. Discussion

In this study, the present inventors further confirmed their previousdata that plasma ALB mRNA is a sensitive marker for liver pathologies.In this study, post-liver transplantation recipients were used as anexample. Twenty-three of the 24 recruited recipients were clinicallystable, had normal biochemical liver function test profile and wereclinically judged to be free from medical or surgical complications atthe time of enrollment. During serial monitoring, it was found that theplasma ALB mRNA concentrations were elevated at the time when clinicaldiagnoses of various complications were made. Among the Unstable group,13 of the 14 documented episodes of complications were associated withelevated ALB mRNA concentrations. In fact, the plasma ALB mRNAconcentrations were elevated before the time the diagnosis was made inthe majority of the episodes. These data further affirm that plasma ALBmRNA is a sensitive marker with utility in the early diagnosis of liverpathologies. For most acute episodes of complications, the plasma ALTactivity-concentrations were not elevated. This observation indicatesthat plasma ALB mRNA is a more sensitive marker for liver pathologiesthan ALT.

On the other hand, elevation in plasma ALB mRNA concentration isspecific to the occurrence of liver pathologies. Majority of themeasurements taken from the Stable group were below the previouslyestablished cutoff value for the detection of liver diseases. Tworecipients in the Stable group had transiently elevated concentrationsof plasma ALB mRNA. These occurrences may be associated with transientliver-associated complications that were not identified clinically.

In summary, elevations in plasma ALB mRNA concentrations allow thesensitive and specific detection of liver pathologies, even at a stagewhen the recipient was symptom-free, the plasma ALTactivity-concentrations were normal and there were no clinical suspicionof complications. Thus, measurement of plasma ALB mRNA concentrationserves as a useful approach for the screening of liver pathologies andregular monitoring of persons who may develop liver-associatedcomplications. As the majority of the liver transplantation recipientsthat were studied in this project had fully recovered from theiroriginal liver disease and the transplantation, the observations thatthe inventors made within this study can be applied to any persons withno prior history of liver disease or had recovered from prior episodesof liver diseases.

Example 3 Elevated Plasma Albumin mRNA Concentration for the Detectionof Fatty Liver Disease

Fatty liver disease, including nonalcoholic fatty liver disease, is themost common chronic liver disease in affluent countries (Farrell et al.,J Gastroenterol Hepatol 2007; 22:775-7. It may progress to cirrhosis andliver cancer. Nonalcoholic fatty liver disease is closely associatedwith metabolic syndrome and central obesity (Wong et al., ClinGastroenterol Hepatol 2006; 4:1154-61).

Ultrasound scan can be used for the diagnosis of fatty liver. However,ultrasound scan is operator dependent, and has limited sensitivity andspecificity for the diagnosis of fatty liver. Liver biopsy andhistological examination provide the definitive proof of fatty liver andany associated hepatic inflammation or fibrosis, but the biopsy is aninvasive procedure and may result in acute complications such ashemorrhage. With advances in technology, it is now possible to useproton magnetic resonance spectroscopy (MRS) as a sensitive andnoninvasive technique to measure hepatic triglyceride (IHTG) content, aindicator of fatty liver (Szczepaniak et al., Am J Physiol EndocrinolMetab 2005; 288:E462-8). However, MRS imaging is time-consuming,requires specialized imaging equipment and trained expert operator. Inthis study the inventors examined whether plasma ALB mRNA is elevated inpatients with fatty liver when compared with healthy controls.

I. Materials and Methods

MRS was performed on apparently healthy volunteers. The IHTG content wasmeasured. Subjects with IHTG content<5% were deemed to have healthylivers while those with IHTG content>5% were diagnosed with fatty liverdisease. In addition, the study also included individuals who werediagnosed to have fatty liver disease by histological examination of aliver biopsy. Peripheral blood was collected from all recruited subjectsinto EDTA blood tubes. The blood samples were processed by the doublecentrifugation protocol as described in the earlier examples to obtainplasma. Plasma ALB mRNA concentration was then measured using theRT-qPCR assay described in the earlier examples.

II. Results

136 volunteers were found by MRS to have IHTG content<5% and thereforewere recruited as normal controls. 47 individuals were found by MRS tohave IHTG content>5% and hence were diagnosed as having fatty liverdisease. 35 individuals were diagnosed with fatty liver disease throughhistological examination of a liver biopsy.

The plasma ALB mRNA concentrations were statistically significantlyhigher among cases with fatty liver disease when compared with thecontrols (Mann-Whitney P<0.001) (FIG. 13). The comparisons were alsostatistically significant if the fatty liver disease cases weresubdivided into those diagnosed by MRS or liver biopsy (Kruskal-Wallisfollowed by pairwise comparisons, P<0.0001 for all pairwise comparisonsbetween the three groups) (FIG. 14). Plasma ALT was measured for thesepatients and it was not elevated in the majority of the cases (FIG. 15).Even in the group diagnosed with fatty liver disease by liver biopsy,46% (16/35) had ALT activity-concentration within the reference cutoff.

III. Discussion

Fatty liver disease is considered an early stage of liver pathologywhich may progress to liver fibrosis, liver cirrhosis and even HCC. Theelevation in plasma ALB mRNA in fatty liver disease patients whencompared with controls indicates that plasma ALB mRNA is indeed a markersensitive enough for the detection of early stage liver pathologies,such as fatty liver disease.

The group diagnosed with fatty liver disease by liver biopsy generallyhas more advanced stage fatty liver disease than those diagnosed by MRS.This is because individuals in the liver biopsy group either havesymptoms or abnormal biochemical liver function test parameters towarrant the referral for the liver biopsy, whereas the MRS groupcomprised of asymptomatic volunteers. The data showed that the groupdiagnosed by liver biopsy had statistically significantly higher plasmaALB mRNA concentrations than the fatty liver disease cases diagnosed byMRS. This observation indicated that plasma ALB mRNA concentrationcorrelates with severity of the liver pathology and can therefore be atool for assessment of the stage of disease, for prognostication and fortreatment monitoring. These data further showed that ALT is a lesssensitive marker than plasma ALB mRNA for the detection of fatty liverdisease and other early stage liver pathologies.

Based on the data obtained for all the presented examples, it has beenconsistently shown that plasma ALB mRNA is a sensitive marker of liverpathologies involving hepatocyte damage, death or apoptosis. Theconcentration of ALB mRNA in plasma becomes elevated when compared tothat of healthy controls for liver pathologies involving hepatocytedamage, death or apoptosis. Such liver or hepatobiliary pathologiesinclude acute conditions, for example acute hepatitis, including thosedue to toxic or viral causes, hypoxic damage, cholangitis and acutebiliary tree obstruction. Such liver or hepatobiliary pathologies alsoinclude chronic conditions, for example, chronic hepatitis, includingdue to hepatitis B or hepatitis C viruses, fatty liver disease, liverfibrosis, liver cirrhosis, hepatocellular carcinoma but not includingthose that recurred after liver transplantation.

The present inventors' data showed that the magnitude of elevation inplasma ALB mRNA compared with healthy controls correlates with severityof the liver pathologies because it is likely to be reflecting thedegree of liver cell damage, death or apoptosis. Thus, the plasma ALBmRNA concentration can be used to prognosticate and monitor theeffectiveness of treatment for all the liver pathologies listed above.

Similarly, elevation in plasma ALB mRNA is shown to be sensitive enoughto detect early stage liver diseases, such as fatty liver disease. Allthe liver pathologies involve hepatocyte damage, death or apoptosis.Therefore, elevated plasma ALB mRNA is also useful as a sensitive markerfor the early stage detection of liver pathologies, for example,progression to active hepatitis in carrier of hepatitis B virus,decompensation or progression of liver cirrhosis, early stagehepatocellular carcinoma but not including those that recurred afterliver transplantation.

All patents, patent applications, and other publications, includingGenBank Accession Numbers, cited in this application are incorporated byreference in the entirety for all purposes.

TABLE 1ALB genotypes of the informative transplantation recipients and donorsPost-transplant Original Post-transplant genotype in Transplant Casegenotype of Genotype genotype in whole blood of type No. recipientof donor plasma of recipient recipient LT L1 A AG AG A LT L4 A AG AG ALT L6 G AG AG G LT L8 AG A A AG LT L9 AG A A — LT L11 A AG AG AG LT L12A AG AG AG LT L18 AG A A AG LT L20 A G G G LT L22 G A A — LT L23 AG A AAG LT L24 AG A A AG LT L25 AG — G AG LT L27 G A A AG LT L29 G AG AG —BMT B1 AG A AG AG BMT B8 A AG A AG BMT B9 AG A AG — BMT B10 AG G AG GBMT B13 AG G AG AG LT, liver transplantation; BMT, bone marrowtransplantation; —, not available. The donor genotype for case L25 wasnot known as the patient underwent the liver transplantation in MainlandChina.

TABLE 2 Profile of the study participants Active CHB Inactive CHB (HBV(HBV DNA ≧10,000 DNA <10,000 Group HCC Cirrhosis copies/mL) copies/mL)Control n 35 25 24 23 207 Sex M (%) 33 (94%) 19 (76%) 20 (83%) 17 (74%)141 (68%) F (%)  2 (6%)  6 (24%)  4 (17%)  6 (26%) 66 (32%) Age (years)55 ± 10 61 ± 9 43 ± 12 47 ± 11 45 ± 10 Albumin (g/L) 42 39 44 48 46(23-48)  (26-47)  (39-50)  (42-49)  (40-52)  Bilirubin 12 24 12 13 13(μmol/L) (6-50)  (3-188) (4-34) (5-25) (2-29) ALP (IU/L) 86 83 70 68 65(43-147) (47-210) (37-111) (40-101) (32-114) ALT (IU/L) 41 40 37 25 21(21-317) (14-197) (12-73)  (10-64)  (10-58)  Hepatitis B surface antigenPositive 29 18 24 23 0 Negative 3 1 0 0 207 Not known 3 4 0 0 0 PlasmaALB 3 454 2 486 3 595 1 642 222 mRNA  (136-57 047)   (0-42 557)   (156-1500 000)  (110-7 847)  (0-2 279) concentration (copies/mL) HCC,hepatocellular carcinoma; CHB, chronic hepatitis B. Age is presented inmean ± SD. Levels of albumin, bilirubin, alkaline phosphatase (ALP),alanine transaminase (ALT) and plasma ALB mRNA concentration arepresented in median (range).

TABLE 3 Primer and probe sequences for ALB SNP genotyping.Primers and probe Sequences SEQ ID NO: Gene-specific primer for5′-TCTTTTGTTGCCTTGGGCTTGT-3′ 1 reverse transcriptionRNA forward PCR primer 5′-ACGTTGGATGCTGAGAAGGAGAGACAAATCAAGAA-3′ 2RNA reverse PCR primer 5′-ACGTTGGATGCTTTTGTTGCCTTGGGCTTG-3′ 3DNA forward PCR primer 5′-ACGTTGGATGTTTCCATTCAAACTCAGTGCACT-3′ 4DNA reverse PCR primer 5′-ACGTTGGATGTGCTCTTTTGTTGCCTTGGG-3′ 5hME extension primer 5′-TTGGGCTTGTGTTTCAC-3′ 6 NB. All PCR primers forSNP genotyping were designed with an addition of a 10-base tag (bold) toavoid interference in mass spectra.

TABLE 4Sequences of primers, probe and calibration standard for ALB mRNA quantification.Primers, probe and synthetic DNA oligonucleotides Sequences SEQ ID NO:ALB forward primer 5′-TCTCTTTAGCTCGGCTTATTCC-3′ 7 ALB reverse primer5′-TCTTTAAACCGATGAGCAACCT-3′ 8 ALB Probe5′-(FAM)CGAGATGCACACAAGAG(MGBNFQ)-3′ 9 ALB calibration standard5′-TTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGA 10 GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTT-3′ NB. FAM is6-carboxyfluorescein, MGB is minor groove binder and NFQ isnon-fluorescent quencher dye.

TABLE 5 Profile of participants with plasma ALT <58 IU/L Active CHBInactive CHB (HBV (HBV DNA ≧10,000 DNA <10,000 Group HCC Cirrhosiscopies/mL) copies/mL) Control N 24 18 20 22 207 Ratio of participantswith 24/35 (69%) 18/25 (72%) 20/24 (83%) 22/23 (96%) 207/207 (100%)normal ALT to all participants (%) Sex M (%) 22 (92%) 14 (78%) 16 (80%)16 (73%) 141 (68%) F (%)  2 (8%)  4 (22%)  4 (28%)  6 (27%)  66 (32%)Age (years) 55 ± 11 58 ± 9 44 ± 12 43 ± 12 45 ± 10 Albumin (g/L) 43 3944 45 46 (24-48)  (26-47)  (40-50)  (42-49) (40-52)  Bilirubin (mmol/L)12 23 12 11 13 (6-50)  (3-188) (4-20)  (5-25) (2-29) ALP (IU/L) 86 82 7163 65 (43-143) (47-136) (40-111)  (40-101) (32-114) ALT (IU/L) 41 32 3527 21 (21-317) (14-58)  (12-56)  (10-53) (10-58)  Hepatitis B surfaceantigen Positive 21 14 20 22 0 Negative 2 1 0 0 207 Not known 1 3 0 0 0Plasma ALB mRNA 3 638 2 167 2 734 1 326 222 concentration (copies/mL) (136-42 675)   (0-23 354)  (156-90 693)  (110-5 542)  (0-2 279) HCC,hepatocellular carcinoma; CHB, chronic hepatitis B. Age is presented inmean ± SD. Levels of albumin, bilirubin, alkaline phosphatase (ALP),alanine transaminase (ALT) and plasma ALB mRNA concentration arepresented in median (range).

TABLE 6 No. weeks from recruitment Plasma ALB to the time mRNA withfirst (copies/mL of ALT level adverse Patient plasma) at (U/L) atoutcome No. recruitment recruitment Subsequent consequence developed 1 023 Well NA 2 320 28 Well NA 3 430 69 Cholangitis; Hepatic ductstrictures 111 4 218 36 Well NA 5 6,696 53 Mild graft rejection followedwith prominent IHD stones 97 and repeated cholangitis 7 719 32 Well NA 825,769 53 Chronic graft rejection confirmed by liver biopsy 60 9 1,657125 Emergency admission with Jaundice; died of septic shock 91 secondaryto spontaneous bacterial peritonitis 11 350 35 Well NA 12 1,209 50Chronic graft rejection with new onset of alcoholic cirrhosis 124 andportal hypertension 13 376 83 Well NA 14 918 32 Well NA 15 1,578 26 9weeks after blood sampling, admitted for anastomotic 9 stenosis withcholangitis. 20 weeks after blood sampling, died of hepatocellularcarcinoma with bone metatasis 17 1,635 48 Bile duct stones and died ofsepsis and ischaemic 58 heart disease 18 632 43 Well NA 19 0 59 Well NA20 1,732 69 Mild fatty liver confirmed by ultrasound; otherwise well NA21 37 26 Well NA 23 100 33 Well NA 24 107 31 Obstructive jaundice 66 25170 28 Well NA 27 55 25 Well NA 28 365 26 Well NA 29 826 10 Well NAPlasma ALB mRNA concentrations greater than 835 copies/mL; elevated ALTactivity concentration (>58 IU/L); and adverse clinical consequence areshown in bold font.

TABLE 7 Table listing the liver-associated complications developed bythe post-liver transplantation recipients in the Unstable group duringthe study period Time of diagnosis (Week No. from the Case time of No.Complications enrollment) U01 Cholangitis with hepatic junction stenosis50 Cholestasis 69 U02 Cholesterolosis with drug-induced like hepaticinjury 66 U03 Cholangitis 25 U04 Post-transplant lymphoproliferativedisease 63 Severe portal gastropathy with gastritis and cholangitis 111U05 Mild rejection with cholangitis 97 Cholangitis with Escherichia coliinfection 108 U06 Budd-chiari syndrome 96 U07 Recurrence of alcoholiccirrhosis with mild 0 chronic rejection U08 Chronic rejection 60 U09 HCVcirrhosis with hepatomegaly 116 U10 Liver abscess 74 Liver fibrosis 107

1-22. (canceled)
 23. A method for monitoring a post-liver transplantpatient, comprising the steps of: (a) determining the concentration ofalbumin mRNA in an acellular blood sample taken from the patient; (b)comparing the concentration of albumin mRNA from step (a) with astandard control; and (c) detecting an increased risk of post-transplantliver complication in the patient when the concentration obtained instep (b) is greater than the standard control, and detecting noincreased risk of post-transplant liver complication in the patient whenthe concentration obtained in step (b) is no greater than the standardcontrol.
 24. The method of claim 23, wherein the patient has normalalanine aminotransferase (ALT) test results.
 25. The method of claim 23,wherein the post-transplant liver complication is not recurringhepatocellular carcinoma (HCC).
 26. The method of claim 23, wherein thepost-transplant liver complication is cirrhosis or rejection.
 27. Themethod of claim 23, wherein the blood sample is plasma.
 28. The methodof claim 23, wherein the blood sample is serum.
 29. The method of claim23, wherein step (a) comprises amplification of the albumin mRNAsequence.
 30. The method of claim 29, wherein the amplification is by apolymerase chain reaction (PCR).
 31. The method of claim 30, wherein thePCR is a reverse transcriptase (RT)-PCR.
 32. The method of claim 30,wherein the PCR is digital PCR.
 33. The method of claim 30, wherein thePCR is real-time quantitative PCR.
 34. The method of claim 23, whereinstep (a) comprises mass spectrometry or hybridization to a microarray,fluorescence probe, or molecular beacon.
 35. The method of claim 23,further comprising repeating steps (a) to (c) at a later time.
 36. Themethod of claim 35, wherein steps (a) to (c) are repeated multipletimes.